Contractile force in muscle fibers is generated by crossbridges that form between the myosin-containing and actin-containing filaments and hydrolyze ATP. During shortening, the two types of filaments slide relative to each other. Using skinned fiber preparations from rabbit, structural changes associated with crossbridge formation and strain were studied by X-ray diffraction and mechanical methods. An electronic, position-sensitive, X-ray detector was characterized for quantitative measurement of two-dimensional diffraction patterns and actin-based layer lines were recorded from rabbit fibers at a sarcomere length of 4.0 mMu. The intensities of reflections from the regulatory protein, tropomyosin, were unaffected by ionic strength, showing that formation of the "low ionic strength" actomyosin crossbridge is not due to a direct effect of low ionic strength on the position of tropomyosin on the actin-containing myofilament. Sarcomere shortening that occurs when rigor (ATP free) fibers are made to expand laterally by decreasing ionic strength and raising pH can generally be accounted for by rotation of the subfragment 2 portion of the actomyosin crossbridge around the backbone of the myosin-containing filament. However, for small lattice expansions an additional source of shortening, which may be related to the physiological force-generating process, appears to be present. Gel electrophoresis showed that loss of both active tension and resting tension in irradiated muscle fibers is caused by degradation of the megadalton proteins, titin and nebulin, and that the smaller muscle proteins (myosin, actin, tropomyosin, troponin, etc.) remain largely intact. Electronmicroscopy showed that titin and/or nebulin in normal fibers act by positioning the myosin-containing filaments in the middle of the sarcomere. The overall goal of the project is to find out how the actomyosin system in muscle fibers produces force and motion, and how the mechanical responses are regulated.